Quantitative Mating Assay

Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology, 1991

1. Grow a culture of the strain whose mating efficiency is to be determined to a density of about 1E7 cells/ml in YEPD. Prepare a similar culture of an appropriate mating type tester strain (PT1{=a}or PT2{=alpha}).

2. Mix 2E6 cells of the experimental strain with 1E7 cells of the tester strain and collect the cells on a 0.45 Ám pore, 25 mm nitrocellulose filter disk. In addition, collect cells of each strain on separate filters (these serve as controls for reversion and/or contamination).

3. Place the filters on the surface of a YEPD plate (cell side up!) and incubate at 30 deg C for 5 hr. 4. Resuspend the cells on each filter in 1 ml of Y-min broth, sonicate for 5-10 sec to disrupt clumps, and dilute in Y-min broth. Plate the cells from the mating mix on Y-min medium to titer diploids. Likewise, plate cells from the single strain filters on SD medium to check for reversion and/or contamination. Calculate the mating efficiency by determining the titer of diploids plus the cells whose mating efficiency is being determined by plating the cells from the mating mix filter on Y-min supplemented with the auxotrophic markers of the experimental strain. The mating efficiency is expressed as the titer of diploids divided by the titer of diploids plus the titer of the experimental strain.